PROJECT SUMMARY The NRF2 transcription factor is the main regulator of cellular redox balance. NRF2 activation in normal cells protects them from environmental insults and prevents diseases. However, NRF2 activation in cancer cells confers chemoresistance and promotes metastasis. Therefore, understanding the direct mechanisms of NRF2 regulation will have broad implications in disease prevention and treatment. To date, much of what we know about the NRF2 pathway arises from piecemeal efforts to identify one regulator at a time. Despite progress in the field, a complete picture of the NRF2 regulatory network is still lacking. This project utilizes a unique combination of CRISPR gene editing technology, flow cytometry based cell sorting, next generation sequencing, and bioinformatics approaches to systematically identify genes that regulate NRF2. We hypothesize that this unique combination of technologies will allow us to systematically unravel the NRF2 regulatory network. We will systematically knockout one gene at a time in cell lines engineered to express a fluorescence protein when NRF2 is activated. By isolating the fluorescing cells through flow cytometry flow sorting and deconvolution of the data through next generation sequencing and bioinformatics, we can identify genes that when knocked out drive NRF2 activation (negative regulators of NRF2) (Aim 1). Using a similar approach we will identify genes that when knocked out will render NRF2 un-inducible by arsenic (Aim2). Upon completion of the proposed studies, we will have identified a vast majority of the genes that regulate NRF2 transcription factor. Methods developed in this project will be readily adaptable include other toxicants and to study other pathways. Findings from the studies will have far reaching implication in our understanding in the cellular adaptive responses to environmental exposure.